SUMMARY Breast cancer is a global healthcare burden, not only for the patients diagnosed with this disease, but also their families and friends. The surgical treatment of breast cancer, while successful, has significant limitations that increase patient anxiety, increase costs, and can increase the risk for local recurrence and lifelong post-operative complications. A primary limitation stems from the lack of an intraoperative microscopic assessment of surgical tumor margins. Our cohesive and productive team with academic, clinical, and industrial representation has successfully developed and demonstrated for the first time the use of intraoperative optical coherence tomography (OCT) for in vivo human imaging of tumor margins during breast cancer surgery using a novel handheld surgical imaging probe. Additionally, the development and use of interferometric synthetic aperture microscopy (ISAM) for in vivo imaging has shown an important improvement in resolution and depth-of-field. Despite these advances, challenges remain for identifying tissue microstructure, particularly between normal fibrous stroma and dense tumor tissue, which are both highly scattering structures. To address these challenges, we propose the novel and innovative application of polarization-sensitive OCT (PS-OCT) and PS-ISAM for intraoperative in vivo imaging in human breast cancer surgery, and hypothesize that these will improve the detection sensitivity and specificity of positive breast tumor margins over standard OCT/ISAM. Realizing that the presence and progression of cancer significantly alters the collagen-based tissue microenvironment, the use of PS-OCT to sensitively detect and quantify birefringence of tissue collagen offers the potential for earlier detection of cancer and the altered microenvironment. By leveraging ISAM and other computational optical image segmentation algorithms, we can more fully characterize the tissue/tumor microenvironment. Through four specific aims, we will implement hardware and innovative software contributions to construct an intraoperative multi-mode system capable of real-time OCT/ISAM and PS-OCT/PS-ISAM, then use this system to investigate the performance of these imaging modes in clinical human studies to determine the sensitivity and specificity of ex vivo and in vivo PS-OCT/PS-ISAM over standard OCT/ISAM, and against the standard-of-care assessments which include post-operative histopathology and intraoperative visual/tactile cues. The successful completion of this project is expected to establish the clinical intraoperative use of these new optical imaging techniques, with the goal of reducing the current unacceptably high reoperation rates in the surgical treatment of breast cancer.